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PROBER identifies proteins associated with programmable sequence-specific DNA in living cells
Smarajit Mondal1, Muthukumar Ramanathan2, Weili Miao1
1Program in Epithelial Biology, Stanford University, Stanford, CA, USA.
Nature Methods
|August 4, 2022
Summary
Researchers developed PROBER, a new method to identify proteins interacting with DNA sequences in cells. This technique enhances signal detection, aiding in understanding gene regulation and disease-associated DNA variants.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA-protein interactions are crucial for gene regulation.
- Alterations in these interactions by DNA variants are linked to polygenic diseases.
- Existing methods for identifying DNA-associated proteins in cells can be slow and have low signal-to-noise ratios.
Purpose of the Study:
- To develop a faster and more sensitive method for identifying and quantifying proteins associated with specific DNA sequences in living cells.
- To improve the signal-to-noise ratio (SNR) in proximity proteomics studies.
- To analyze how DNA variants and mutations affect protein-DNA associations.
Main Methods:
- Developed Proximal Biotinylation by Episomal Recruitment (PROBER).
- PROBER utilizes high-copy episomes to amplify the signal.
- Employs proximity proteomics (BioID) to identify interacting proteins with DNA sequences of interest.
Main Results:
- PROBER successfully quantified constitutive and inducible associations of transcription factors and chromatin regulators with target DNA sequences.
- The method identified binding quantitative trait loci influenced by single-nucleotide variants.
- PROBER revealed altered regulator associations at the hTERT promoter due to cancer hotspot mutations, indicating increased binding of specific gene activators.
Conclusions:
- PROBER offers a rapid and sensitive approach for identifying proteins bound to specific DNA sequences in living cells.
- The method can effectively detect changes in protein-DNA interactions caused by genetic variations and mutations.
- PROBER facilitates the study of gene regulation and the molecular basis of diseases linked to DNA variants.
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